Lyophilized trypsin was reconstituted immediately prior to usage in cold H2O to give a final concentration of either 0.02 or 0.1 μg/μL. To validate trypsin activity, 1 μL of Cytochrome C (CytC, 1 mg/mL) was spotted on each slide [30 (link)]. Trypsin was applied using a SunCollect sprayer (SunChrom, Friedrichsdorf, Germany) or an HTX M5 Sprayer (HTX‐Technologies, Chapel Hill, North Carolina, USA), abbreviated “HTX sprayer” throughout the paper, using customized and optimized spraying protocols for each sprayer (technical details in Supplementary Table S1 ). After trypsin application, the slides were incubated in a humidity chamber for 2 h or 17 h at 50°C or 37°C, respectively. To keep humidity constant, the chamber was filled with either 50% (v/v) methanol or saturated aqueous K2SO4 solution. Thereafter, trypsin digestion was terminated by matrix application. The applied trypsin density (W) was 1.3 and 6.7 ng/mm2 when using 0.02 and 0.1 μg/μL trypsin solutions with the HTX sprayer, and 3.3 and 16.7 ng/mm2 when using 0.02 and 0.1 μg/μL trypsin solutions with the SunCollect sprayer. Trypsin density was calculated according to Supplementary Equation S1 .
Htx m5 sprayer
Manufactured by HTX Technologies
Sourced in United States
The HTX M5 sprayer is a versatile laboratory equipment designed for a variety of applications. It features a high-performance spray nozzle and can be used to apply liquids or suspensions with precision.
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Lab products found in correlation
Ti2 epifluorescence microscope, by Nikon (1 mentions)
Cryostat, by Leica (1 mentions)
Solarix 7 t ft icr mass spectrometer, by Bruker (1 mentions)
Pannoramic desk 2 dw scanner, by 3DHISTECH (1 mentions)
Maldi tof tof mass spectrometer, by Bruker (1 mentions)
Maldi source, by Bruker (1 mentions)
Timstof flex, by Bruker (1 mentions)
Cm1860 cryostat, by Leica (1 mentions)
11 protocols using htx m5 sprayer
1
Optimization of Trypsin Digestion for MALDI-MSI
2
3D Tumor Spheroid Characterization by MALDI-MSI
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Spheroids were embedded in 10% gelatin, snap frozen and stored at −80 °C. Upon analysis, serial sections were taken at 10 µm using a Leica cryostat. Bright-field and immunofluorescent (IF) images of the spheroids were acquired using a Ti2-Epi Fluorescence Microscope (Nikon) prior to mass spectrometry imaging. Matrix deposition was carried out using the HTX M5 sprayer (HTX Technologies LLC, Chapel Hill, NC, USA). Then, 9-aminoacridine (9AA) at a concentration of 10 mg/mL was deposited onto the spheroid sections using flow rate of 160 µL/min and a temperature of 60 °C for a total of 14 passes using the crisscross setting. MS images were acquired using a Bruker SolariX 7T FT-ICR mass spectrometer (Bruker Daltonics, Billerica, MA, USA) equipped with a dual ESI/MALDI ion source and with a smartbeam II Nd:YAG laser, 355 nm. Data were acquired using the small laser setting and the oversampling method [36 (link)], which resulted in a pixel size/image resolution of ~20 µm. The instrument was operated in the negative ion mode over the mass range m/z 150–3000. Following data acquisition, the slides were stained with hematoxylin and eosin (H&E) and scanned using the Pannoramic DESK II DW scanner (3DHISTECH, Budapest, Hungary).
3
MALDI Matrix Application Protocol
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The MALDI matrix 2,5-dihydroxybenzoic acid (DHB) was prepared to a concentration of 30 mg/mL in 70% methanol. Matrix was applied using an HTX-M5 Sprayer (HTX Technologies, Chapel Hill, NC), with a spray spacing of 2.5 mm at a temperature of 75 °C using a flow rate of 100 μL/min. The distance of the sprayer nozzle was 50 mm from the sample and a spray pressure of 10 psi with a spray nozzle motion velocity of 1200 mm/min was used. Four passes were used to apply MALDI matrix.
4
MALDI Matrix Application Protocol for Brain and Pancreatic Samples
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The MALDI matrix 2,5-dihydroxybenzoic acid (DHB) was prepared to a concentration of 30 mg ml−1 in 70% methanol for brain samples. For pancreatic samples, DHB was prepared at a concentration of 10 mg ml−1 in 50% ethanol. The matrix was applied using an HTX-M5 Sprayer (HTX Technologies), with a spray spacing of 2.5 mm at a temperature of 75 °C using a flow rate of 100 μl min−1. The distance of the sprayer nozzle was 50 mm from the sample, and a spray pressure of 10 psi with a spray nozzle motion velocity of 1,200 mm min−1 was used. Four passes were used to apply the MALDI matrix.
5
MALDI-TOF/TOF Analysis of Single Cells
6
MALDI Imaging of Metabolites in Tissue
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Before MALDI analysis, matrix coating (either 12 or 14 passes) was applied to the desiccated tissue sections by using an HTX M5 sprayer (HTX Technologies) with 10 mg mL−1N-(1-Naphthyl) ethylenediamine dihydrochloride (NEDC; Sigma), dissolved in 70:30 methanol:water. The spraying parameters were set as follows: 80 °C nozzle temperature, 0.1 mL min−1 flow rate, 1000 mm min−1 velocity, 2 mm track spacing, 10 psi pressure, 3 L min−1 gas flow rate, and 10 s drying time for each pass.
MALDI samples were analyzed by using a timsTOF fleX with a MALDI source (Bruker Daltonics). All MALDI images were collected in negative polarity in MS1 mode at 50 μm pixel size. The laser was set as a single focused beam with a laser power of 85%, a laser frequency of 10 kHz, and 200 shots per pixel. The mass range was 50-400 m/z. Mass calibrations were performed by using 1 mM sodium formate solution. AMP, stearate, and palmitate were analyzed from sections with 12 layers of matrix coating, while NAA was analyzed in sections with 14 layers applied.
MALDI samples were analyzed by using a timsTOF fleX with a MALDI source (Bruker Daltonics). All MALDI images were collected in negative polarity in MS1 mode at 50 μm pixel size. The laser was set as a single focused beam with a laser power of 85%, a laser frequency of 10 kHz, and 200 shots per pixel. The mass range was 50-400 m/z. Mass calibrations were performed by using 1 mM sodium formate solution. AMP, stearate, and palmitate were analyzed from sections with 12 layers of matrix coating, while NAA was analyzed in sections with 14 layers applied.
7
MALDI-TOF Sample Preparation Protocol
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After enzymatic digestion, samples
were dried and alpha-Cyano-4-hydroxycinnamic acid (7 mg/mL in 50%
acetonitrile and 1% trifluoroacetic acid) spiked with a 1.67 μg/mL
[Glu]-Fibrinopeptide B internal standard was applied using an HTX
M5 Sprayer (HTX Technologies, LLC) with the following parameters:
79 °C nozzle temperature, 10 passes, 7 mg/mL concentration, 1300
velocity, 0.07 mL/min flow rate, 2.5 mm track spacing, CC pattern,
10 psi nitrogen pressure, 3 L/min gas flow rate, 0 drying, 40 mm Nozzle
height. The target plate was dried and subsequently dipped twice in
cold (4 °C) aqueous 5 mM ammonium phosphate and dried in a desiccator
until mass spectrometry data acquisition.40 (link),41 (link)
were dried and alpha-Cyano-4-hydroxycinnamic acid (7 mg/mL in 50%
acetonitrile and 1% trifluoroacetic acid) spiked with a 1.67 μg/mL
[Glu]-Fibrinopeptide B internal standard was applied using an HTX
M5 Sprayer (HTX Technologies, LLC) with the following parameters:
79 °C nozzle temperature, 10 passes, 7 mg/mL concentration, 1300
velocity, 0.07 mL/min flow rate, 2.5 mm track spacing, CC pattern,
10 psi nitrogen pressure, 3 L/min gas flow rate, 0 drying, 40 mm Nozzle
height. The target plate was dried and subsequently dipped twice in
cold (4 °C) aqueous 5 mM ammonium phosphate and dried in a desiccator
until mass spectrometry data acquisition.40 (link),41 (link)
8
Collagen Digestion by Sprayed Collagenase
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Activity-characterized
collagenase (0.1 mg/mL) was resuspended in ammonium bicarbonate (10
mM) and calcium chloride (1 mM) buffer solution (pH 7.35) and applied
using an HTX M5 Sprayer (HTX Technologies, LLC) with the following
parameters: 40 °C nozzle temperature, 15 passes, 0.1 mg/mL concentration,
0.025 mL/min flow rate, 3 mm track spacing, CC pattern, 10 psi nitrogen
pressure, 3 l/min gas flow rate, 0 drying, 40 mm Nozzle height. The
target plate was transferred to a humidity chamber where collagen
fragments were digested at 37 °C for 5 h.
collagenase (0.1 mg/mL) was resuspended in ammonium bicarbonate (10
mM) and calcium chloride (1 mM) buffer solution (pH 7.35) and applied
using an HTX M5 Sprayer (HTX Technologies, LLC) with the following
parameters: 40 °C nozzle temperature, 15 passes, 0.1 mg/mL concentration,
0.025 mL/min flow rate, 3 mm track spacing, CC pattern, 10 psi nitrogen
pressure, 3 l/min gas flow rate, 0 drying, 40 mm Nozzle height. The
target plate was transferred to a humidity chamber where collagen
fragments were digested at 37 °C for 5 h.
9
MALDI Imaging of Zebrafish Cryosections
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Zebrafish embryos were placed in a 10 mm × 10 mm × 5 mm biopsy cryomold (Ted Pella, Redding, CA) and embedded in Thermo Scientific Shandon M1 embedding media (Thermo Fisher Scientific, Waltham, MA). After freezing at −20 °C, the block was sectioned at 10 μm thickness at −16 °C and thaw-mounted onto cleaned indium tin oxide (ITO) slides (Delta Technologies, Loveland, CO). All cryosectioning was done on a Leica CM1860 cryostat (Buffalo Grove, IL). A traditional organic matrix was prepared at 10 mg/mL DHB in 50% methanol/50% water and sprayed using an HTX M5 sprayer (HTX Technologies, Chapel Hill, NC) with a nozzle temperature of 85 °C, using 8 spray passes at a flow rate of 0.075 mL/min with no drying time. Gold nanoparticles (AuNPs) were sprayed at either 30 or 45 °C, with one pass at a flow rate of 0.010 mL/min with 2 s drying time. One ITO slide was used for all experiments, with parafilm used to mask different sections of the slide so that multiple spray compositions were contained on this one slide. The final slide layout and description of regions of interest are shown in Fig. 2 .![]()
Optical image of imaged slide with regions of interest highlighted and sample preparation conditions defined.
10
Thiosalicylic Acid Spray Application
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Thiosalicylic acid (TSA) was applied using an HTX M5 Sprayer (HTX Technologies, Chapel Hill, USA). The spray solution was prepared as follows: 154 mg TSA were weighed into a glass beaker and suspended in 3 mL water. By adding 1 M NaOH, the suspension was brought to pH = 10 and then filled up to 5 mL. Under these alkaline conditions, TSA is completely dissolved. The solution is finally made up to 10 mL with methanol and loaded into the sample loop of the sprayer. 20 passes were sprayed onto the sample surface with a flow rate of 50 µL/min, a spray nozzle velocity of 750 mm/min and 50 °C nozzle temperature. Track spacing was set to 2 mm in a criss-cross (CC) scan-pattern.
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